The Roles And Molecular Mechanism Of Transcription Factor FOXM1 In Hydatidiform Mole

I.ObjectiveGestational trophoblastic disease(GTD)refers to a group of pregnancy-related disorders,ranging from the pre-malignant forms of partial and complete hydatidiform mole(HM),to the malignant forms of invasive mole,choriocarcinoma,placental-site trophoblastic tumor(PSTT)and epithelioid trophoblastic tumor(ETT).The malignant forms of GTD are termed gestational trophoblastic neoplasia(GTN).Statistics shows that about 9-20% of hydatidiform moles are deteriorating to gestational trophoblastic neoplasia.GTN is a unique human tumor because it is related to the heredity of fetal tissue.As a key factor in cancer research in recent years,FOXM1 inhibitors,such as salinomycin A and thiostrepton have been confirmed to inhibit tumor cells proliferation,migration and invasion by regulating the expression of FOXM1.Forkhead box M1(FOXM1)belongs to the Fox transcription factor family,is an important regulator in animal development of cell proliferation,migration,invasion and angiogenesis.In recent years,FOXM1 has been found to be highly expressed in many solid tumors such as lung cancer,pancreatic cancer,gastric cancer and hepatocellular carcinoma in many studies.In the process of embryonic development and the stage of oncology,FOXM1 promotes the transformation of G1/S and G2/M by regulating a variety of downstream factors,thus promoting cell proliferation.That isparticularly critical in the stage of oncology.Another study shown that FOXM1 b can promote the angiogenesis,invasion and migration process of oncology by binding the promoter region of Vescular endothelia growth factor(VEGF).This study investigated the role of FOXM1 in the pathogenesis of HM.FOXM1 protein expressions in clinical samples of endometrium,term placentas and HMs were determined by immunohistochemistry as well as in cell lines were detected by western blot and Immunofluorescence staining.The effects of FOXM1 on JAR cell proliferation,migration and invasion were assessed by corresponding functional assays.The effect of AKT and ERK signaling pathways on the expression of FOXM1 were investigated by treating JAR cells with the AKT inhibitor LY294002 or the ERK inhibitor U0126 respectively.Indeed,the effect of AKT and ERK signaling pathways in JAR cells were assessed by corresponding functional assays.Our results demonstrated that FOXM1,regulated by AKT and ERK signaling pathways,was invested in the pathogenesis of HM,promoting proliferation,migration and invasion in JAR cells.1.To investigate the clinic pathological relationship of FOXM1 in hydatidiform moles2.To obtain the expression levels and location of FOXM1 in human trophoblast cells lines;3.To determine the affect of FOXM1 overexpression and knockdown on proliferation,migration and invasion in JAR cells.4.To determine the affect of AKT and ERk on FOXM1 expression in JAR cells.5.To examine the molecular mechanism of FOXM1 in hydatidiform moles.II.Methods1.The study of transcription factor FOXM1 in the hydatidiform mole proliferation mechanism;1)The collection of clinical tissues included endometrium,term placenta and hydatidiform moles;2)The expression and location of FOXM1,FOXO1 and FOXO3 a in hydatidiformmole,placenta and endometrium were detected by immunohistochemistry;3)The expression of FOXM1 in human choriocarcinoma cell lines Be Wo and JAR were detected by Western Blot.The localization of FOXM1 were detected by immunofluorescence assay.4)The recombinant FOXM1 overexpression plasmid and recombinant FOXM1 interference plasmid(si RNA)were transfected into human choriocarcinoma cell lines JAR by lipofection method.The expression of FOXM1 was detected by Western Blot;5)The effect of FOXM1 in JAR cells proliferation abilities were detected by CCK-8assay.The effect of FOXM1 in JAR cells migration and invasion abilities were detected by transwell assay;2.Study on the regulation of AKT,ERK signaling pathways for FOXM1 in hydatidiform mole1)The collection of clinical tissues included endometrium,term placenta and hydatidiform moles;2)The expression and location of AKT and ERK in endometrium,term placenta and hydatidiform mole tissues were detected by immunohistochemical;3)JAR cells were treated with different concentrations of AKT signaling pathway inhibitor LY294002 and ERK signaling pathway inhibitor U0126 to detect these regulation to FOXM1 by Western Bolt;4)The invasion and migration abilities were evaluated by transwell assay shown that the affect of FOXM1 in cell migration and invasion.The cell ability of cell proliferation was detected by CCK8 assay;The FOXM1 expression and location were detected by immunofluorescence;5)The affects of AKT signaling pathway inhibitor LY294002 and ERK signaling pathway inhibitor U0126 on cell migration and invasion were detected by transwell assay,the affacts of AKT signaling pathway inhibitor LY294002 and ERK signaling pathway inhibitor U0126 on cell proliferation were detected by CCK8 assay;The downstream factors expression such as MMPs and VEGF weredecteced by Western Blot;III.Results1.The study of transcription factor FOXM1 in the hydatidiform mole proliferation mechanism;1)FOXM1 was highly expressed in hydatidiform mole tissues,mainly located in trophoblast,cytotrophoblast and syncytiotrophoblast.The trend of FOXO1 and FOXO3 a expression were opposite to FOXM1;2)By Western blot,FOXM1 expression in Be Wo was higher than that in JAR cells;Immunofluorescence showed that FOXM1 mainly located in cytoplasm in JAR cells while FOXM1 were located both cytoplasm and nucleus in Be Wo cells;3)JAR cells transfected with FOXM1 plasmids,the expression of FOXM1 was highly increased.JAR cells transfected with si RNA,the expression of FOXM1 was decreased;4)FOXM1 overexpression/knockdown promotes/attenuates JAR cells proliferation,migration and invasion;5)FOXM1 overexpression/knockdown promotes/attenuates the expression of VEGF and MMPs in JAR cells.2.Study on the regulation of AKT,ERK signaling pathways for FOXM1 in hydatidiform mole1)AKT and ERK were highly expressed in hydatidiform mole tissues and located in cytoplasm and nucleus of cytotrophoblast,syncytiotrophoblast and villus trophoblast;2)AKT signaling pathway inhibitor,LY294002 and ERK signaling pathway inhibitor,U0126,reduced the expression of FOXM1 in a dose-dependent manner;3)AKT and ERK signaling pathways affect the proliferation,migration and invasion by regulating the expression of FOXM1 in JAR cells;4)AKT and ERK signaling pathways reduced the expression of MMPs and VEGF by regulating the expression of FOXM1.IV.Conclusions1.The study of transcription factor FOXM1 in the hydatidiform mole proliferation mechanism1)FOXM1 is highly expressed in hydatidiform mole tissues and mainly located in cytoplasm of trophoblast,cytotrophoblast and syncytiotrophoblast;2)FOXM1 affects cell proliferation in human choriocarcinoma cells;3)FOXM1 affects cell migration and invasion in human choriocarcinoma cells;4)FOXM1 regualted the expression of MMPs and VEGF in human choriocarcinoma cells.2.Study on the regulation of AKT,ERK signaling pathways for FOXM1 in hydatidiform mole1)AKT and ERK signaling pathways were activated in hydatidiform mole tissues and mainly located in the nucleus and cytoplasm of villus trophoblast,cytotrophoblast and syncytiotrophoblast;2)AKT and ERK signaling pathways regulate the expression of FOXM1;3)AKT and ERK signaling pathways affect cell proliferation,migration and invasion by regulating the expression of FOXM1;4)AKT and ERK signaling pathways affect MMPs and VEGF expression by regulating the expression of FOXM1;...